Phage display technology, i.e., the use of filamentous phage to display recombinant proteins and peptides, is well known and used for selecting proteins and peptides with desired functions or improved characteristics from complex libraries. Phage display is widely used for the isolation of human antibodies through clonal selection of antibody fragments in prokaryotic host systems. Filamentous phage from the Ff group, including M13, fl and fd phage, are commonly used. Vectors capable of directing the generation of recombinant phage and phagemid expressing fusions of viral coat proteins with proteins of interest in E. coli have been developed and are widely available. There are two broad categories of vectors used for phage display: phage and phagemid.
When proteins are displayed on phage, the gene encoding the recombinant display protein is included in the phage genome. As a result, all phage particles display the recombinant protein and contain only the recombinant phage genome. In the case of phagemid, the recombinant protein is encoded as a fusion with the g3p on a plasmid (phagemid) which also contains the filamentous phage packaging signal. Bacteria carrying such phagemids make large amounts of the recombinant display protein, but are unable to make phage unless the bacteria carrying the phagemid also contain helper phage, which supply all the other proteins required to make functional phage.
Helper phages1, 2 are essentially normal Ff phages with a number of modifications: their packaging signal is severely disabled, they contain an additional origin of replication, and they usually carry antibiotic resistance genes. The disabled packaging signal does not prevent the helper phage from making phage particles when alone in a bacterium, but in the presence of a phagemid, which has an optimal packaging signal, the phagemid should be packaged in preference to the helper phage. As a result, phagemid preparations are both phenotypically and genotypically heterogeneous (FIG. 1). Accordingly, the displayed protein may be either wild type (derived from the helper phage) or recombinant (derived from the phagemid), and the packaged genome may be either phage or phagemid (see FIG. 1).
The different antibiotic resistance genes carried by phage and phagemid allow one to select for bacteria that contain both the phagemid and the helper phage. While the disabled packaging signal in helper phage should significantly reduce the presence of helper phage in any phagemid preparation, helper phage can sometimes be present at levels equal to, or exceeding, phagemid levels. This can significantly compromise subsequent selections.
Phagemid and phage libraries differ in a number of practical ways, and in general, the use of phagemids provides several advantages. At the DNA level (preparing DNA, cloning, transfection efficiency), phagemids are easier to work with, and as a result, phagemid libraries can be made far larger than phage libraries. It is also easier to produce soluble proteins in phagemids when an amber stop codon is inserted between the displayed protein and g3p3. Although soluble protein could theoretically be made in phage display libraries using a similar genetic arrangement, the low copy number of the vector and the weakness of the g3p promoter and ribosome binding site, results in levels of soluble protein which are too low for most practical purposes, requiring recloning into expression vectors4. Another advantage of phagemids concerns the relative resistance to deletions of extraneous genetic material. Filamentous phage vectors, in general, have a tendency to delete unneeded DNA, as a result of the selective growth advantage a smaller phage has over a larger one. Phagemids suffer far less from this disadvantage and as a result are more stable.
However, phage libraries have considerable operational advantages. To amplify phage libraries it is sufficient to grow bacteria, and phage are produced. There is no need to add helper phage, and subsequent antibiotics, at specific optical densities. This makes phage far easier to use in selections, and also makes automation far more straightforward. Furthermore, the genetic and phenotypic homogeneity of phage libraries eliminate the possibility of helper phage overgrowth. As each phage particle in a phage library displays up to five copies of the displayed protein (using a g3p display system), and only 1-10% of phage particles in a phagemid library display a single copy of the displayed protein5, antibodies selected from phage libraries tend to be more diverse, but have lower average affinities, a result of the avidity effects caused by the display of multiple proteins per phage particle4.
Phagemid display, by virtue of the display of single proteins, results in the selection of fewer unique binders, which tend to have higher affinities4. For similar reasons, affinity maturation6-9 can only be carried out with phagemid vectors.
Recently, a number of groups2, 10-14 have attempted to combine some of the advantages of phage and phagemids by creating helper phages deleted or mutated in gene 3. Initial experiments involved the creation of g3p deleted helper phage, packaged in bacterial strains expressing gene 3 in trans. These allowed higher display levels, but suffered from the problem that when p3 was derived from plasmids, but not the E. coli chromosome12, such plasmids could also be packaged, albeit at low levels2, and that helper phage titers tend to be very low. More recently, conditional g3p deletions have been created by the introduction of suppressible stop codons in g311, 13, allowing production of helper phage in suppressor strains, and the packaging of phagemids in non-suppressor strains, where the helper phage is unable to make its own g3p. An alternative method14 involves a helper phage which has part of g3 deleted (CT helper phage). This deleted p3 can incorporate into phagemid particles, but because it lacks the N1/2 domains is unable to participate in infection, with the net result that phagemid not displaying the recombinant g3p fusion protein are unable to propagate. In practice, although not in theory, this is similar to a modified helper phage system15 in which a trypsin site is introduced within the helper phage g3p. When trypsin is used for elution, all phagemid particles containing only helper phage g3p are inactivated and prevented from infecting and subsequent propagation. Both systems result in a lower background during selection. These different helper phage systems are compared in Table 1.
Although it is difficult to compare the different systems, those containing suppressible stop codons in g311, 13 appear to be most effective, given that they produce helper phage, and phagemid, titers, as high as, or almost as high as, standard helper phage. Although these systems may overcome some of the disadvantages of helper phage, they do not avoid one of the main problems associated with the use of helper phage: the need to make helper phage and add it to growing bacterial cultures at relatively restricted phases of the growth cycle.